Influence of Different Extraction Methods on the Changes in Bioactive Compound Composition and Antioxidant Properties of Solid-State Fermented Coffee Husk Extracts

In brewing coffee, a huge amount of food waste is generated; that waste, coffee husks in particular, should be comprehensively exploited. They offer a rich source of bioactive compounds such as caffeine, chlorogenic acid, and trigonelline. The aim of this study was to investigate the effects of extraction methods on the bioactive compounds and antioxidant activity of such waste. Coffee husks in this study were fermented with S. cerevisiae based on a solid-state fermentation technique. The study method included ethanolic or water extraction with varied controllable factors, i.e., temperature (60, 100°C) and extraction technique. Bioactive contents were investigated with the Folin–Ciocalteu assay and 1H-NMR spectroscopy. The antioxidant activity was investigated with DPPH and FRAP assays. Results show that yields were the highest in the extract of fermented coffee husks at 100°C. The highest levels of bioactive contents (total trigonelline content at 3.59% and antioxidant activity at 23.35% (DPPH) and 25.9% (FRAP)) were found in the ethanolic extract of fermented coffee husks at 60°C. The bioactive content and bioactivity, including antioxidant activity, depended on different raw materials, preparation methods, and extraction conditions. This study illustrates the potential for using food waste such as coffee husks as a sustainable source of bioactive compounds or bioactive extracts.


Introduction
Cofee is one of the most popular nonalcoholic beverages in the world.It has a unique aroma, favor, and taste, which result from a complex combination of more than 1,000 chemical components [1], although the predominant bioactive compounds in cofee are cafeine, cafeic acid, chlorogenic acids, trigonelline, diterpenes, and melanoidins, many of which have been scientifcally proven to promote or improve health.In fact, cofee itself has many reported health benefts.It is recognized as a health drink when consumed in appropriate amounts [2].In addition to the expected health benefts of drinking cofee, cofee is a crop that is important to the global economy.It is cultivated in more than 70 countries worldwide; the global cofee market in 2022 was valued at 503.86 billion USD and is expected to reach 797.12 billion USD by 2030 [3].In 2021, the estimated global consumption of cofee beans was approximately 10.25 million metric tons, and demand is generally increasing every year [4].However, cofee production also produces an incredible amount of biological waste since the most commonly consumed part of cofee is the bean; the remainder ends up as food waste that results from processing, accounting for approximately 8.45 million metric tons or around 45.2% of the total weight of cofee fruit.Te waste product includes the skin, pulp, mucilage, parchment, and silver bean [5].Cofee husks ofer great potential to decrease the industry's carbon footprint while providing value to consumers via upcycled food products.Some of these have a history of being consumed as traditional foods or beverages (pulp), while some parts (e.g., husk and silver bean) have been studied to assess their safety and benefts as novel foods.Previous studies have found that waste products from cofee processing ofer a source of nutrients, dietary fbers, and bioactive compounds that are not inferior to the cofee beans themselves [6,7].However, it has been reported that the disposal of cofee waste products can have severe ecotoxicological efects.Cofee grounds can reduce the levels of oxygen in the water and earth, resulting in the death of local fauna due to lack of oxygen or an increasingly acidic environment.In addition, this waste can emit 28.6 million tons of carbon dioxide per year and results in the release of methane into the atmosphere, exacerbating the climate crisis [8,9].
Te primary solid waste product from cofee processing is cofee husk, which contains both nutrients and nonnutrients, including carbohydrates (35-85%), proteins (5-11%), fat (0.3-5.5%), dietary fber (24-43%), minerals (K, Ca, Mg, Zn, Cu, and Fe), polyphenol, favonoid, and other bioactive compounds [10,11].Te prominent bioactive compounds found in cofee husks are cafeine, chlorogenic acid, and trigonelline [12].Chlorogenic acid is an important bioactive polyphenol that is responsible for the bitter and astringent taste of cofee.It is one of the most abundant phenolic acids and is found in green cofee beans rather than roasted cofee beans [13].Trigonelline is a plant alkaloid formed by a methylation reaction of niacin (vitamin B3).Previous review articles have concluded that chlorogenic acid and trigonelline have pharmacological activities (e.g., antioxidant, anti-infammatory, antimutagenic, antitumor, antidiabetic, antiobesity, antihypertension, hepatoprotective, nephroprotective, cardioprotective, and neuroprotective) and thus play a role in reducing the risk of various diseases [14].Solid-state fermentation is a process that involves the cultivation of microorganisms on a solid matrix (raw materials) in conditions that are without water or nearly free water.Previous studies have reported that the solid-state fermentation process can increase the content and bioactivity of bioactive compounds.It has been found that solid-state fermentation of cofee grounds can increase the total favonoid, total phenolic, chlorogenic acid, quinic acid, and cafeic acid content [15][16][17].Te process may ofer the potential to develop functional ingredients for health benefts in the future.However, some bioactive compounds, including chlorogenic acid and trigonelline, are unstable and easily degraded, especially when exposed to heat or light [18].In addition, there are alternative extraction methods that use sustainable source materials or environmentfriendly solvents (i.e., ethanol and water) and deliver an extract rich in bioactive compounds; these methods seem promising, and it will be necessary to study their usefulness for reducing environmental problems and promoting sustainable development [19].Terefore, the present study aimed to investigate the efect of optimal and simple green extraction of bioactive compounds from fermented cofee husks [20].In addition, utilizing cofee husk, which is a food waste product from cofee processing, is one way to reduce the amount of food waste and decrease its negative impact on the environment [20].Tis complies with sustainable development goals in goal 12: responsible consumption and production and goal 13: climate action [21].

Materials and Methods
2.1.Chemicals and Raw Materials.Ethanol, sulfuric acid, and phosphoric acid, all of which were of analytical grade from Honeywell (Seelze, Germany), were used in the process.Sodium metabisulfte, used to stabilize the chlorogenic acids during the assays, was of analytical grade and was purchased from Sigma Aldrich (Steinheim, Germany).Chlorogenic acid was purchased from Biopurify (Chengdu, China).Trigonelline, sodium 3-(trimethylsilyl)-[2,2,3,3-d4]-1-propionate (TSP), deuterium oxide, and other chemicals used were purchased from Merck Millipore (Burlington, Massachusetts, USA).Cofee husks from the wet depulping and demucilaging process of cofee beans (Cofea arabica) were cleaned until water was clear.Raw cofee husk was split into lots of 3 kg and kept in a freezer at −20 degrees Celsius prior to experiments; batches of 1 kg of cofee husk were thawed at room temperature when needed.

Preparation of Fermented Cofee Husk.
Fermented cofee husk was used in this study.It was prepared by a solid-state fermentation method adapted from a previous study [22].Arabica cofee husk was thawed from freezing at −20 degrees Celsius to room temperature and then blended with a Termomix TM31 multifunction disintegrator (Vorwerk, Wuppertal, Germany) at maximum speed for 1 minute.Ground cofee husk was fermented with 10 ml activated yeast (1 g of S. cerevisiae) in a sealed bag at room temperature for 8 hours in the dark, with a solid-to-solvent ratio of 0.1 g/ ml.After the fermentation period, 0.5% sodium metabisulfte (Na 2 S 2 O 5 ) was added to inactivate yeast, and the mixture was kept in a freezer at −20 degrees Celsius until ready for use in the study.

Experimental Design.
Five diferent extraction methods were designed and tested in this study: (I) extraction from fermented cofee husk by distilled water at 100 degrees Celsius for 20 minutes, (II) extraction from fermented cofee husk by distilled water at 60 degrees Celsius for 20 minutes, (III) extraction from fresh cofee husk by distilled water at 60 degrees Celsius for 20 minutes, (IV) extraction from fermented cofee husk by ethanol, using a refux technique at 60 degrees Celsius for 20 minutes, and (V) extraction from fermented cofee husk soaked with ethanol inside a closed container at room temperature for 6 days.All extraction methods were performed with a solid-to-solvent ratio of 0.5 g/ml.After extraction periods, the liquid was fltered from the cofee husk.Te extract was then dried in a hot air oven at 60 degrees Celsius until the samples were completely 2 Te Scientifc World Journal dry (for ethanolic extracts, ethanol was evaporated in a rotary evaporator before drying).Te weights of all samples were recorded, and the percentage of yield was calculated.Each sample was stored in an airtight container in a dark and dry place at 25 degrees Celsius until it was ready for analysis.

Determination of Total Phenolic
Contents.Total phenolic content was analyzed using the Folin-Ciocalteu reagent adapted from a previous study [23].Te extracts were mixed with saturated sodium bicarbonate and 10% Folin-Ciocalteu reagent (v/v).Tis solution was then incubated in the dark at room temperature for 30 minutes.After that, the absorbance was measured at a wavelength of 765 nm by using a DU-8800D spectrophotometer (QTECH, Russia).Te total phenolic content was presented as gallic acid equivalents (GAE) in milligrams per gram of sample extract (mg•GAE/g).

Determination of Antioxidant Activities.
Antioxidant assays were performed using ferric ion reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging by a colorimetric assay adapted from a previous study [24,25].For the FRAP assay, the extracts were mixed with FRAP reagent that was freshly prepared by mixing 300 mM acetate bufer (pH 3.6), 20 mM FeCl 3 •6H 2 O solution, and 10 mM 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ in 40 mM HCl) at the ratio of 10 : 1 : 1. Te mixture was incubated for 4 minutes at room temperature before measuring the absorbance at the wavelength of 593 nm by using a DU-8800D spectrophotometer (QTECH, Russia).Te electron donating capacity of ferric ion (Fe 3+ ) was presented as a ferrous ion (Fe 2+ ) equivalent in millimolars per gram of sample extract or percent inhibition by comparison with the standard curve of ferrous sulfate (FeSO 4 ).Te DPPH assay mixture consisted of the extracts and DPPH solution (250 μg/ml of DPPH in ethanol).It was placed into a 96 wellplate and incubated in the dark at room temperature for 30 minutes.After that, the absorbance was measured at the wavelength of 517 nm by using an RT-2100C microplate reader and SoftMax Pro Software analysis software (Meditech, Australia).Te free radical scavenging capacity was presented as a percent inhibition calculated from the absorbance loss of DPPH radicals.Ascorbic acid was used as the standard for this assay, and the extract concentrations were in the range of the ascorbic acid standard curve.

Results and Discussion
3.1.Percentage of Extraction Yields.Extraction yields from each extraction method were presented in percentages that were calculated from the ratio of extract weights to cofee husk weight (shown in Table 1).Te extract yield was found to be the highest in the sample that used water extraction of fermented cofee husk at 100 degrees Celsius (method I: 5.1%) and the lowest in the sample that macerated fermented cofee husk in ethanol for 6 days (method V: 0.8%).It was found that, at the same temperature, the extraction yield by the water-solvent method was higher than the ethanolsolvent method.However, in the same extraction solvent, increasing the temperature resulted in a higher extraction yield.At least one previous study had reported that the type of extraction solvent and temperature were important factors that afected the efciency of plant extraction [20].In fact, the solvent has a great infuence on the yield of the extract, and it determines the type of components that can be extracted and dissolved in the extract.Tis is because of diferences in the polarity of the extraction solvents, which have an efect on the solubility of the components in the raw material [26].Generally, heating is expected to increase the extraction efciency because the solubility of compounds should increase with the rise in temperature [27].In addition, this study also demonstrated that under the same extraction conditions, fermented cofee husk yielded less than unfermented cofee husk.Furthermore, when using the same extraction method (refux technique with ethanol), the extraction yield in this study (1.1%) was lower than the extraction yield in previous studies (4.8-27.58%)[28,29], which may have resulted from the use of diferent raw materials for extraction (fermented or fresh cofee husk).Consistently, previous studies have found that the type of raw material was one of the factors that infuenced the extraction yield as did other extraction factors, i.e., extraction time, solid-to-solvent ratio, and pH value of the solvent [20,30] It would be valuable to carry out further studies aimed at understanding the relationships between the extraction factors in order to increase the extraction efciency.
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Total Phenolic Contents.
All cofee husk extracts exhibited total phenolic contents in the range of 0.0002-0.0082mg•GAE/g dry weight of extract (shown in Table 1).Te extracts obtained using ethanol extraction tended to exhibit the highest total phenolic contents.Tis method provided total phenolic contents up to 20-fold higher than water extraction.According to the results, water extraction seemed to be less efective for the extraction of phenolics.Tis result is consistent with a previous study that reported a tendency for total phenolic and total favonoid contents to increase with an increase in ethanol concentration in the extraction solvent [31].Tis may be a result of the polarity of the extraction solvent having an efect on the solubility of compounds.Te solubility of phenol compounds in the extraction solvent is a very infuential factor on the total phenolic contents of extracts.Previous studies reported that the total phenolic content of cofee husk extracts was in the range of 0.0118-4.5459mg•GAE/g, depending on diferent raw materials and extraction conditions [32,33].Tus, the extraction efciency is dependent on the selection of the optimal solvent, temperatures, and mechanical agitation for use in the extraction process [34].However, it has been reported that the use of mixed solvents, e.g., water-ethanol can extract more bioactive compounds than pure solvents [20,35,36].However, reducing the solvent volume through reuse can be difcult when using mixed solvents, especially in small and medium industries.Ethanol can form a binary azeotrope with water in mixed solvents [37].As a result, the water cannot be completely removed using simple distillation techniques.Terefore, it is difcult to recycle ethanol when using mixed solvents.To obtain pure ethanol for reuse, the ternary system technique required a substantial quantity of energy [37,38].Previous studies reported that sequential extractions (from nonpolar to polar solvents) can improve the extraction efciency and uncovering of widely phytochemical compounds and bioactivity [39,40].However, the use of a wide variety of extraction solvents required proper waste management.Tis involved costs, logistics, laws, practices, storage capacity, safety considerations, and technology available in the production area, when scaled up from laboratory to industry scale [41].Consequently, we designed the study using only pure solvents (water or ethanol) so that it can be scaled up to a small industry scale, which is the limitation of this work.

Antioxidant Activities.
Oxidation is a very complicated mechanism, so a single method cannot be relied on to completely evaluate the antioxidant properties.In this study, the antioxidant properties of cofee husk extracts were determined by DPPH and FRAP assays, and the results are shown in Table 2. Te antioxidant properties from both assays were found to be the highest in the sample that used water extraction of fermented cofee husk at 100 degrees Celsius (method III), followed by the sample that used ethanol-refux extraction of fermented cofee husk at 60 degrees Celsius (method IV).Te lowest levels of antioxidant properties were found in the sample that used water extraction of fermented cofee husk at 60 degrees Celsius (method II).In general, bioactive compounds are present in the bound form with the matrix; thus, the high temperature was able to break down interactions between the bioactive compounds and the matrix [42].As a result, antioxidant properties were increased in samples that were extracted under higher temperature.However, the rise in temperature may decrease the bioactivity of the extract because; at the hightemperature condition, some bioactive compounds could sufer degradation or a loss of function [20].From the results, fresh cofee husk extract was found to have higher antioxidant properties than fermented cofee husk extract.Tis may be the result of yeast metabolism that can decompose or change some components of the raw material during the fermentation process.Consistently, previous studies have identifed the efects of fermentation on various components (including phenolics).Specifcally, fermentation can reduce the contents or change the structure of bioactive compounds such as catechin content or lead to the conversion of sinapine to sinapic acid [43][44][45].However, the results with respect to the antioxidant properties were not the same as those of total phenolic content, which indicates that the cofee husk extracts also contain other compound groups that have antioxidant properties in addition to phenolic acid.Further studies on the phytochemical profle of the cofee husk extracts are needed.In addition, the antioxidant property assay that was carried out in this study is based on the electron transfer (ET) mechanism only.Terefore, further studies with assays based on the hydrogen atom transfer mechanism should be performed to more concisely evaluate antioxidant properties.4 Te Scientifc World Journal

NMR Analysis of Chlorogenic Acid and Trigonelline.
Te 1 H NMR spectra proposed as fngerprints are shown in Figure 1.Te chlorogenic acid peak in the chemical shift region from 5.3 to 7.7 ppm stands out.Te results indicate that a chlorogenic acid peak can be identifed in the extraction from fresh cofee husks, while it is rarely detected in extracts from fermented cofee husks.Tis contradicts a previous study that reported that solid-state fermentation may increase chlorogenic acid content by up to 400% [22], perhaps due to a result of free water content generated in the process of thawing frozen cofee husk from −20 degree Celsius to room temperature.Water is one of the necessary factors for yeast activity.Its presence may have resulted in increased fermentation and reproduction of yeast in the solid-state fermentation processes, which would be consistent with a previous study that found that chlorogenic acid content in cofee beans decreased after refermentation processes [46].In addition, the water content in the fermentation system was an important factor afecting the regulation of chlorogenic acid degradation kinetics.Te presence of water in the process results in the conversion of chlorogenic acid to cafeic acid through a hydrolysis reaction [47].In the result of the 1 H NMR analysis, a peak of trigonelline was found to stand out in the chemical shift region from 8.0 to 9.2 ppm.Trigonelline is the major alkaloid found in cofee.A previous study reported that trigonelline content was signifcantly decreased during the fermentation period [16].However, with respect to the fermentation time, this study was shorter (8 hours) than that of the previous studies (24-168 hours) [16,48].Tis is one factor that may have led to diferent amounts of trigonelline.Tus, a quantitative investigation of trigonelline is required.

Total Trigonelline Content.
A quantitative assessment for the determination of trigonelline in cofee husk extract was performed on the 1 H NMR signal of trigonelline H-2 (9.2 ppm, singlet).Calibration curves were employed to calculate the concentrations of trigonelline in the extracts, which were found to be in the range of 2.46-3.87%(w/w)  Te Scientifc World Journal (shown in Figure 2), and were the highest in the sample that used ethanol-refux extraction of fermented cofee husk at 60 degrees Celsius (method IV).Tis indicates that cofee husks are a good source of trigonelline when compared to cofee beans (around 1% w/w) [49].In fact, trigonelline is highly soluble in water and hot ethanol [50].However, from the results, trigonelline was found to be higher in the ethanolic extract than in the aquatic extract.Tis may be the result of water contained in the cofee husk sample.Consistently, previous studies have reported that the presence of water in the extraction solvent causes the plant cells to swell, allowing easier difusion of solvent into the plant cells.In addition, the polarity of the solvent can be adjusted to be highly suitable for the dissolution of trigonelline [51,52].In the same extraction solvent, we found that an increase in extraction temperature did not have any efect on the trigonelline content due to the fact that trigonelline is a heat-stable alkaloid that does not undergo thermal degradation during the extraction process.However, it may not be possible to extract all the trigonelline from the matrix at 100 °C for 20 min [53].It is possible that the trigonelline content of the extract in this study was lower than the actual trigonelline content in the cofee husk.In this study, we did not fnd a signifcant diference between fresh and fermented cofee husks.

Conclusion
Tis study demonstrates that cofee husk obtained as food waste from cofee processing ofers a potential renewable plant source for bioactive extracts.Cofee husk extracts exhibit antioxidant properties based on an electron transfer mechanism and contain high levels of trigonelline content compared to cofee that is generally consumed.Te study found that the optimal extraction method relied on ethanolrefux extraction of fermented cofee husk at 60 degrees Celsius, which provided an extraction yield of 1.1%, total phenolic content of 0.008 mg•GAE/g, and total trigonelline content of 3.59%.Te antioxidant activity of this extract as measured by the DPPH assay and FRAP assay was measured at 23.35% and 25.79%, respectively.Tis study did not fnd a diference between fresh and fermented cofee husk extracts.However, it was found that the condition of sample preparation and extraction factors (including temperature, type of solvent, extraction technique, and extraction time) had a signifcant efect on antioxidant properties and bioactive compound content.In particular, the water content is an important factor in the chlorogenic acid content which control the hydrolysis of cafeic acid.It would be worthwhile to conduct further studies to understand the relationships between the extraction factors in order to increase the effciency of the process.Te fndings of this study are the efect of the extraction methods on the antioxidant activity and the content of bioactive compounds, especially trigonelline.Tis may lead to the development of the extraction method with high health benefts for cofee husk extracts as antioxidants, anti-infammatory, antiaging agents, etc.However, although cofee husk extract has drawn interest as a functional ingredient for potential health benefts, further studies should be conducted to assess its potential for further utilization.Figure 2: Total trigonelline content (% w/w) of water extract of fermented cofee husk at 100 °C (FCW100), water extract of fresh cofee husk at 60 °C (NFCW60), ethanolic extract of fermented cofee husk by the refux technique at 60 °C (FCE60), and ethanolic extract of fermented cofee husk by the maceration technique for 6 days (FCES6).Te statistical diferences were analyzed by using one-way ANOVA followed by the Tukey post hoc test.ns, * , * * , * * * , and * * * * mean p > 0.05, ≤0.05, <0.01, <0.001, and <0.0001, respectively.

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Figure 1 : 1 H
Figure1: 1 H NMR fngerprints of (a) chlorogenic acid standard; (b) fermented cofee husk was extracted with water at 60 °C; (c) fermented cofee husk was extracted with water at 100 °C; (d) fermented cofee husk was extracted with ethanol at 60 °C; (e) fermented cofee husk was soaked with ethanol; (f ) fresh cofee husk was extracted with water at 60 °C; (g) fresh ground cofee husk.

Table 1 :
Extraction yields and total phenolic content of the lyophilized samples of cofee husks by each extraction method.SD (n ≥ 3).Diferent superscript letters, p ≤ 0.05; same letter (p > 0.05), obtained from ANOVA with the Tukey post hoc test.